Sandbox 37

LYSOZYME
Lysozyme, pictured to the right, is an enzyme found commonly in saliva, tears, mucus, and even egg whites. This particular enzyme breaks down bonds of bacterial cell walls. Specifically, lysozyme serves to catalyze the hydrolysis of the Beta (1->4) glycosidic linkage which occurs between N-acetylmuramic acid and N-acetylglucosamine (NAG) of peptidoglycan. Lysozyme is also capable of hydrolyzing polyNAG which constitutes chitin, a major component of fungi cell walls as well as the exoskeletons of insects and crustaceans. .

The Lysozyme in egg white, specifically hen egg whites (HEW), is the most widely studied and hence the most widely understood species of the enzyme. The picture to the right reveals a HEW lysozyme's cartoon structure with the secondary structure in grey and the yellow being salt bridges throughout the molecule.



= History = Laschtschenko first discovered the acitivty of the enzyme in chicken eggs in 1909. It was not until 1922 when Alexander Fleming stumbled upon accidental observations concerning lysozyme from nasal drippings. He was working in a lab and found that a bacterial plated petri dish which nasal drippings had dripped into resulted in disrupted bacterial activity. He continued by intesively studying the mechanism by which this occurred and found the cleavage sites of lysozyme. Since this time, Lysozyme has become a commercially available enzyme and is even useful in the treatment of ulcers and some bacterial infections. .

Mechanism
The mechanism by which lysozyme cleaves beta linkages and be viewed here: ]].

As is seen here, lysozyme works through a hydrolysis of a glycosidic bond. The fourth sugar which is bound to the lysozyme is put through torsional strain and eventually put into the half chair conformation which greatly weakens the strength of the bond between the fourth and fifth sugars. A proton from Glutamic acid 35 to the Oxygen in the neighboring sugar results in the cleavege of the bond and results in an oxonium ion. Aspartic acid 52 has a carboxylate groups which serves to stablize the ion and acts as a nucleophile to connect to the fourth sugar of the original six sugar chain. Water then attacks the site which results in water being present on the carbon where the bond was originall formed. Glutamine 35 assists in general base catalysis in order to attach the water to the molecule. .

Inhibitors and Activators
It has been determined that Lysozyme's activity is able to be both activated and inhibited by a variety of agents. The enzyme is activated by EDTA while it is inhibited by SDS, most alcohols, N-acetyle-D-glucosamine, and a variety of oxidizing agents. These inhibitors work by either competitive inhibition of the active site (N-acetyle-D-glucosamine) or by interfering with the mechanism of action (Alcohols and Oxidizing agents).

= Structure =

Lysozyme contains 129 amino acids which are labelled in this presentation of lysozyme. The molecular weight of lysozyme is 14.7 kDa. The strucutre was disovered in 1965 via the use of X-ray crystallography by Chilton Phillips. It should be noted that lysozyme is very easy to crystallize and as such the purification of the enzyme is quite simple and effective.

Secondary Structure
The secondary structure of lysozyme consists of 5 beta pleated sheets along with 5 alpha helices. These structures are displayed as cartoon rockets where the alpha helices are labelled pink and the beta pleated sheets are yellow. The two shortest cartoon rockets which should be yellow beta pleated sheets are labelled pink by the program when told to label different secondary structures differently. This is due to the fact that two of the beta pleated sheets are slightly abnormally shaped and thus can be confused for alpha helices.



Disulfide Bonds
Found throughout the structure of lysozyme are disulfide bonds which are labelled yellow and cysteine residues which are labelled blue. Seen here are 8 cysteine residues along with 4 disulfide bonds. The bonds are an integral part of the strucutre and stability of lysozyme. .

Hydrogen Bonds
Hydrogen bond interactions are seen in light blue (the yellow represents the disulfide bonds). Hydrogen bonding occurs between proton donors and acceptors of specific residues. In this way, hydrogen bonds greatly impact the overall structure and stability of the lysozyme molecule as a whole.

= Distribution of Polar and NonPolar residues =

A distribution of polar vs nonpolar residues can be viewed HERE. The residues on this backbone stucture of lysozyme are labelled blue if they are polar and red if they are nonpolar. By viewing this structure one is capable of seeing the overall dispersion of polar vs. nonpolar residues. Polar residues, which are hydrophillic, are generally found on the outside of a molecule so that they can interact with surrounding water molecules. This is much more apparent when viewing THIS structural format (blue again represents polar while red represents nonpolar residues. Lysozyme is slightly unique in its polar vs nonpolar residues in that, as is apparent in the structure just mentioned, there are still a large number of nonpolar residues towards the outside surface of the molecule. This is due to the fact that lysozyme is a relatively "open" molecule meaning that it is not tightly bound and thus does not hide all of its nonpolar residues with an outward polar residue shell.

= Ligand =

In biochemistry, a ligand is considered any substance which is able to bind to a biomolecule, in this case an enzyme, and form a complex which serves a biological purpose, in this case the hydrolysis of a glycosidic bond.

The ligand of lysozyme is thus N-acetylmuramic acid and N-acetylglucosamine which bind to the active site of lysozyme very specifically. Lysozyme is only capable of binding polysaccharide chains six sugars in length. The enzyme binds to the fourth sugar and distorts it into a a half chair conformation which results in the glycosidic bond becoming quite weak.

In the binding site of lysozyme are the residues glutamic acid 35 (purple) and aspartate 52 (green). In this structure, the surface of the protein is labelled yellow so that the cleft can be clearly seen. The cartoon structure can be seen here with the same residues visible again. This structure reveals the active site in a more relative sense for the protein as it is difficult to view precisely where the outward surface of the protein is.

It was also determined that asp101, trp62, trp63, asn59, ala107, val109 and gln57 all play a role in binding of the ligand to the binding site.

Studies and Applications of Lysozyme
A study recently performed by Saeed A. Khan revealed that lysozyme can be beneficial in the protection against intentional exposure to anthrax which is a topic of particular concern considering terrorists threats made in the past. The study was performed in such a way that the anthrax causing microbe was inserted into HEW and then the activity was monitored. Khan reported that "based on our results, it looks like that lysozyme could be used to either slow down or prevent the growth of an avirulent form of the bacterium Bacillus anthracis that causes anthrax," however, "more research is needed on other types of foods, including ground beef, milk, fruit juices, and vegetables." This was in reference to the fact that they also attempted to insert the microbe into milk and ground beef with similar benefical results concerning the inhibition of the microbe causing anthrax.

=References=